Preparation of soy protein isolate using calcium chloride extraction (“S703 cip”)

ABSTRACT

A soy protein product having a protein content of at least about 60 wt % (N×6.25) d.b., preferably an isolate having a protein content of at least about 90 wt % (N×6.25) d.b., is formed by a procedure in which soy protein is extracted from a soy source material using an aqueous calcium chloride solution at low pH, generally about 1.5 to about 5, and separating the resulting aqueous soy protein solution from residual soy protein source. The resulting clarified aqueous soy protein solution may be diluted and the pH adjusted within the range of 1.5-5.0. The solution may be concentrated by ultrafiltration, diafiltered and then dried to provide the soy protein product. Alternatively, the concentrated and optionally diafiltered soy protein solution may be optionally adjusted in pH within the range of 1.5-7.0 then diluted into water to cause the formation of a precipitate, separating the precipitate from the diluting water (supernatant) and drying the separated soy protein to form a soy protein product having a protein content of at least about 60 wt % (N×6.25) d.b., preferably a soy protein isolate having a protein content of at least about 90 wt % (N×6.25) d.b. The supernatant may be processed to form soy protein products having a protein content of at least about 60 wt % (N×6.25) d.b., preferably a soy protein isolate having a protein content of at least 90 wt % (N×6.25) d.b. Alternatively, the precipitate from the dilution step may be re-solubilized in the diluting water by adjustment of the pH to resolubilize the precipitate and form a protein solution. The soy protein solution may be concentrated while maintaining the ionic strength substantially constant by using a selective membrane technique followed by optional diafiltration and drying. The soy protein product is soluble in acidic medium and produces transparent, heat stable solutions and hence may be used for protein fortification of soft drinks and sports drinks.

REFERENCE TO RELATED APPLICATION

This application is a U.S. National Phase filing under 35 USC 371 ofPCT/CA2012/000443 filed May 9, 2012 claiming priority under 35 USC119(a) from U.S. patent application Ser. No. 13/067,201 (now U.S. Pat.No. 8,404,299) filed May 17, 2011 which itself is a continuation-in-partof U.S. patent application Ser. No. 12/828,212 (now U.S. Pat. No.8,501,265) filed Jun. 30, 2010 which itself claims priority under 35 USC119(e) from U.S. Provisional Patent Application No. 61/213,647 filedJun. 30, 2009.

FIELD OF INVENTION

The present invention is concerned with the preparation of soy proteinproducts.

BACKGROUND TO THE INVENTION

In U.S. patent applications Ser. No. 12/603,087 (7865-415) filed Oct.21, 2009 (US Patent Publication No. 2010-0098818) and Ser. No.12/923,897 (7865-454) filed Oct. 13, 2010 (US Patent Publication No.2011-0038993), assigned to the assignee hereof and the disclosures ofwhich are incorporated herein by reference, there is described thepreparation of a soy protein product, preferably a soy protein isolate,which is completely soluble and is capable of providing transparent andheat stable solutions at low pH values. This soy protein product may beused for protein fortification of, in particular, soft drinks and sportsdrinks, as well as other acidic aqueous systems, without precipitationof protein. The soy protein product is produced by extracting a soyprotein source with aqueous calcium chloride solution at natural pH,optionally diluting the resulting aqueous soy protein solution,adjusting the pH of the aqueous soy protein solution to a pH of about1.5 to about 4.4, preferably about 2.0 to about 4.0, to produce anacidified clear soy protein solution, which may be optionallyconcentrated and/or diafiltered before drying.

SUMMARY OF THE INVENTION

It has now been surprisingly found that a soy protein product having aprotein content of at least about 60 wt % (N×6.25) d.b. may be formed bya procedure involving extraction of the soy protein source with calciumchloride at low pH values.

In one aspect of the present invention, a soy protein source material isextracted with aqueous calcium chloride solution at low pH and theresulting aqueous soy protein solution is optionally diluted, optionallyadjusted in pH within the acidic range, then subjected toultrafiltration and optional diafiltration to provide a concentrated andoptionally diafiltered soy protein solution, which may be dried toprovide the soy protein product.

In another aspect of the present invention, a soy protein sourcematerial is extracted with aqueous calcium chloride solution at low pHand the resulting aqueous soy protein solution is optionally diluted,optionally adjusted in pH within the acidic range, then subjected toultrafiltration and optional diafiltration to provide a concentrated andoptionally diafiltered soy protein solution. The concentrated andoptionally diafiltered soy protein solution may then be optionallyadjusted in pH within the pH range of about 1.5 to about 7, preferablyabout 4 to about 7, more preferably about 5 to about 7 and diluted withwater to fractionate the soy proteins into a precipitate rich inglobulins and a supernatant rich in albumin proteins and containingtrypsin inhibitors. Precipitate formed by the dilution step may becollected and further processed or dried as is to provide the soyprotein product, but with a reduced level of trypsin inhibitors.

In another aspect of the present invention, the concentrated andoptionally diafiltered and optionally pH adjusted soy protein solution,prepared as described above is diluted into water. The pH of the dilutedsample is adjusted to about 1.5 to about 4.4, preferably about 2.0 toabout 4.0 to re-solubilize protein precipitated by the dilution step.The diluted and pH adjusted solution may then be optionally heat treatedand/or concentrated and/or diafiltered.

The soy protein products provided herein, having a protein content of atleast about 60 wt % (N×6.25) d.b., are soluble at acid pH values toprovide transparent and heat stable aqueous solutions thereof. The soyprotein products may be used for protein fortification of, inparticular, soft drinks and sports drinks, as well as other aqueoussystems without precipitation of protein. The soy protein product ispreferably an isolate having a protein content of at least about 90 wt%, preferably at least about 100 wt % (N×6.25) d.b.

In accordance with one aspect of the present invention, there isprovided a method of producing a soy protein product having a soyprotein content of at least about 60 wt % (N×6.25), on a dry weightbasis, which comprises:

-   -   (a) extracting a soy protein source with aqueous calcium salt        solution, generally calcium chloride solution, at low pH,        generally about 1.5 to about 5.0, to cause solubilization of soy        protein from the protein source and to form an aqueous soy        protein solution,    -   (b) at least partially separating the aqueous soy protein        solution from residual soy protein source,    -   (c) optionally diluting the aqueous soy protein solution,    -   (d) optionally adjusting the pH of the aqueous protein solution        to a value within the range of about 1.5 to about 5.0,        preferably about 1.5 to about 4.4, more preferably about 2.0 to        about 4.0, and differing from the pH of extraction,    -   (e) optionally polishing the aqueous soy protein solution to        remove residual particulates,    -   (f) optionally concentrating the aqueous soy protein solution        while maintaining the ionic strength substantially constant by        using a selective membrane technique,    -   (g) optionally diafiltering the concentrated soy protein        solution, and    -   (h) optionally drying the concentrated and diafiltered soy        protein solution.

The soy protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

A variation of this procedure may be adopted to produce the product witha reduced content of albumin proteins and trypsin inhibitors. In such avariation, the concentrated and optionally diafiltered soy proteinsolution is optionally adjusted in pH within the range of about 1.5 toabout 7.0, preferably about 4.0 to about 7.0, more preferably about 5.0to about 7.0, then diluted into water to yield a precipitate with areduced content of albumin proteins and trypsin inhibitors. Theprecipitate may be collected and dried to yield the product or theprecipitate may be solubilized in water at pH about 1.5 to about 4.4,preferably about 2.0 to about 4.0 and then dried. Alternatively, thesolution formed by solubilizing the precipitate in water at pH about 1.5to about 4.4, preferably about 2.0 to about 4.0 may be optionally heattreated and/or polished and/or concentrated and/or diafiltered beforedrying.

Accordingly, in another aspect of the present invention, there isdescribed a method of producing a soy protein product having a soyprotein content of at least about 60 wt % (N×6.25), dry weight basis,which comprises:

-   -   (a) extracting a soy protein source with aqueous calcium salt        solution, generally calcium chloride solution, at low pH,        generally about 1.5 to about 5.0, to cause solubilization of soy        protein from the protein source and to form an aqueous soy        protein solution,    -   (b) at least partially separating the aqueous soy protein        solution from residual soy protein source,    -   (c) optionally diluting the aqueous soy protein solution,    -   (d) optionally adjusting the pH of the aqueous protein solution        to a value within the range of about 1.5 to about 5.0,        preferably about 1.5 to about 4.4, more preferably about 2.0 to        about 4.0, and differing from the pH of extraction,    -   (e) optionally polishing the aqueous soy protein solution to        remove residual particulates,    -   (f) concentrating the aqueous soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (g) optionally diafiltering the concentrated soy protein        solution,    -   (h) optionally adjusting the pH of the concentrated and        optionally diafiltered soy protein solution to a value within        the range of about 1.5 to about 7.0, preferably about 4.0 to        about 7.0, more preferably about 5.0 to about 7.0,    -   (i) diluting the concentrated and optionally diafiltered and pH        adjusted soy protein solution into water,    -   (j) separating precipitate formed from the diluting water,        termed the supernatant, and    -   (k) drying the separated soy protein precipitate.

The soy protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

Another variation of this procedure may be adopted to produce theproduct. In such a variation, the concentrated and optionallydiafiltered and optionally pH adjusted soy protein solution is dilutedinto water and the pH adjusted after dilution, which re-solubilizesprecipitate formed by the dilution step. The resulting pH adjustedsolution is optionally heat treated and/or polished and/or concentratedand/or diafiltered before drying to yield the product.

Accordingly, in a further aspect of the present invention, there isdescribed a method of producing a soy protein product having a soyprotein content of at least about 60 wt % (N×6.25), dry weight basis,which comprises:

-   -   (a) extracting a soy protein source with aqueous calcium salt        solution, generally calcium chloride solution, at low pH,        generally about 1.5 to about 5.0, to cause solubilization of soy        protein from the protein source and to form an aqueous soy        protein solution,    -   (b) at least partially separating the aqueous soy protein        solution from residual soy protein source,    -   (c) optionally diluting the aqueous soy protein solution,    -   (d) optionally adjusting the pH of the aqueous protein solution        to a value within the range of about 1.5 to about 5.0,        preferably about 1.5 to about 4.4, more preferably about 2.0 to        about 4.0, and differing from the pH of extraction,    -   (e) optionally polishing the aqueous soy protein solution to        remove residual particulates,    -   (f) concentrating the aqueous soy protein solution while        maintaining the ionic strength substantially constant by using a        selective membrane technique,    -   (g) optionally diafiltering the concentrated soy protein        solution,    -   (h) optionally adjusting the pH of the concentrated and        optionally diafiltered soy protein solution to a value within        the range of about 1.5 to about 7.0, preferably about 4.0 to        about 7.0, more preferably about 5.0 to about 7.0,    -   (i) diluting the concentrated and optionally diafiltered and pH        adjusted soy protein solution into water,    -   (j) adjusting the pH of the diluted sample to a value within the        range of about 1.5 to about 4.4, preferably about 2.0 to about        4.0 to re-solubilize protein precipitate formed by the dilution        step,    -   (k) optionally concentrating the pH adjusted soy protein        solution while maintaining the ionic strength substantially        constant by using a selective membrane technique,    -   (l) optionally diafiltering the concentrated, pH adjusted soy        protein solution, and    -   (m) drying the concentrated and optionally diafiltered, pH        adjusted soy protein solution.

The soy protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

Although this specification refers mainly to the production of a soyprotein isolate, the concentration and/or diafiltration steps describedherein may be manipulated to produce a soy protein product of lesserpurity, for example, a soy protein concentrate having a protein contentof at least about 60 wt %, but which has substantially similarproperties to the isolate.

The novel soy protein products of the invention can be blended withpowdered drinks for the formation of aqueous soft drinks or sportsdrinks by dissolving the same in water. Such blend may be a powderedbeverage.

The soy protein products provided herein may be provided as an aqueoussolution thereof having a high degree of clarity at acid pH values andwhich is heat stable at these pH values.

In another aspect of the present invention, there is provided an aqueoussolution of the soy product provided herein which is heat stable at lowpH. The aqueous solution may be a beverage, which may be a clearbeverage in which the soy protein product is completely soluble andtransparent or an opaque beverage in which the soy protein product doesnot increase the opacity. The soy protein product also has goodsolubility at about pH 7. An aqueous solution of the soy proteinproduct, prepared at a near neutral pH, such as a pH of about 6 to about8, may be a beverage.

The soy protein products produced according to the process herein lackthe characteristic beany flavour of soy protein isolates and aresuitable, not only for protein fortification of acidic media, but may beused in a wide variety of conventional applications of protein isolates,including but not limited to protein fortification of processed foodsand beverages, emulsification of oils, as a body former in baked goodsand foaming agent in products which entrap gases. In addition, the soyprotein product may be formed into protein fibers, useful in meatanalogs, and may be used as an egg white substitute or extender in foodproducts where egg white is used as a binder. The soy protein productmay also be used in nutritional supplements. Other uses of the soyprotein product are in pet foods, animal feed and in industrial andcosmetic applications and in personal care products.

GENERAL DESCRIPTION OF INVENTION

The initial step of the process of providing the soy protein productinvolves solubilizing soy protein from a soy protein source. The soyprotein source may be soybeans or any soy product or by-product derivedfrom the processing of soybeans including but not limited to soy meal,soy flakes, soy grits and soy flour. The soy protein source may be usedin the full fat form, partially defatted form or fully defatted form.Where the soy protein source contains an appreciable amount of fat, anoil-removal step generally is required during the process. The soyprotein recovered from the soy protein source may be the proteinnaturally occurring in soybean or the proteinaceous material may be aprotein modified by genetic manipulation but possessing characteristichydrophobic and polar properties of the natural protein.

Protein solubilization from the soy protein source material is effectedmost conveniently using calcium chloride solution, although solutions ofother calcium salts may be used. In addition, other alkaline earth metalcompounds may be used, such as magnesium salts. Further, extraction ofthe soy protein from the soy protein source may be effected usingcalcium salt solution in combination with another salt solution such assodium chloride. Additionally, extraction of the soy protein from thesoy protein source may be effected using water or other salt solution,such as sodium chloride, with calcium chloride subsequently being addedto the aqueous soy protein solution produced in the extraction step.Precipitate formed upon addition of the calcium chloride then is removedprior to subsequent processing.

As the concentration of the calcium salt solution increases, the degreeof solubilization of protein from the soy protein source initiallyincreases until a maximum value is achieved. Any subsequent increase insalt concentration does not increase the total protein solubilized. Theconcentration of calcium salt solution which causes maximum proteinsolubilization varies depending on the salt concerned. It is usuallypreferred to utilize a concentration value less than about 1.0 M, and,more preferably, a value of about 0.10 M to about 0.15 M.

In a batch process, the solubilization of the protein is effected at atemperature of from about 1° C. to about 100° C., preferably about 15°to about 65° C., more preferably about 20° C. to about 35° C.,preferably accompanied by agitation to decrease the solubilization time,which is usually about 1 to about 60 minutes. It is preferred to effectthe solubilization to extract substantially as much protein from the soyprotein source as is practicable, so as to provide an overall highproduct yield.

In a continuous process, the extraction of the soy protein from the soyprotein source is carried out in any manner consistent with effecting acontinuous extraction of soy protein from the soy protein source. In oneembodiment, the soy protein source is continuously mixed with calciumsalt solution and the mixture is conveyed through a pipe or conduithaving a length and at a flow rate for a residence time sufficient toeffect the desired extraction in accordance with the parametersdescribed herein. In such a continuous procedure, the solubilizationstep is effected rapidly, in a time of up to about 10 minutes,preferably to effect solubilization to extract substantially as muchprotein from the soy protein source as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 1° C. and about 100° C., preferably about 15° C. to about65° C., more preferably between about 20° C. and about 35° C.

The extraction is generally conducted at a pH of about 1.5 to about 5.0.The pH of the extraction system (soy protein source and calcium saltsolution) may be adjusted to any desired value within the range of about1.5 to about 5.0 for the extraction step by the use of any convenientfood grade acid, usually hydrochloric acid or phosphoric acid.

The concentration of soy protein source in the calcium salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein extraction step with the aqueous calcium salt solution hasthe additional effect of solubilizing fats which may be present in thesoy protein source, which then results in the fats being present in theaqueous phase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 50 g/L, preferably about 10 toabout 50 g/L.

The aqueous calcium salt solution may contain an antioxidant. Theantioxidant may be any convenient antioxidant, such as sodium sulfite orascorbic acid. The quantity of antioxidant employed may vary from about0.01 to about 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit the oxidation of any phenolics in theprotein solution.

The aqueous phase resulting from the extraction step then may beseparated from the residual soy protein source, in any convenientmanner, such as by employing a decanter centrifuge or any suitablesieve, followed by disc centrifugation and/or filtration, to removeresidual soy protein source material. The separated residual soy proteinsource may be dried for disposal. Alternatively, the separated residualsoy protein source may be processed to recover some residual protein.The separated residual soy protein source may be re-extracted with freshcalcium salt solution, with the re-extraction conducted in the pH rangeof about 1.5 to about 5.0, and the protein solution yielded uponclarification combined with the initial protein solution for furtherprocessing as described below. Alternatively, the separated residual soyprotein source may be processed by a conventional isoelectricprecipitation procedure or any other convenient procedure to recoversuch residual protein.

Where the soy protein source contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein. Alternatively, defatting of the separatedaqueous protein solution may be achieved by any other convenientprocedure.

The aqueous soy protein solution may be treated with an adsorbent, suchas powdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the separated aqueous protein solution. For powderedactivated carbon, an amount of about 0.025% to about 5% w/v, preferablyabout 0.05% to about 2% w/v, is employed. The adsorbing agent may beremoved from the soy protein solution by any convenient means, such asby filtration.

The resulting aqueous soy protein solution may be diluted generally withabout 0.5 to about 10 volumes, preferably about 0.5 to about 2 volumesof aqueous diluent, in order to decrease the conductivity of the aqueoussoy protein solution to a value of generally below about 90 mS,preferably about 4 to about 31 mS. Such dilution is usually effectedusing water, although dilute salt solution, such as sodium chloride orcalcium chloride, having a conductivity of up to about 3 mS, may beused.

The diluent with which the soy protein solution is mixed may have atemperature of about 2° to about 70° C., preferably about 15° to about65° C., more preferably about 20° to about 35° C.

The optionally diluted soy protein solution may be adjusted in pH to avalue different from the extraction pH but still within the range ofabout 1.5 to about 5.0, preferably about 1.5 to about 4.4, morepreferably about 2.0 to about 4.0, by the addition of any suitable foodgrade acid, such as hydrochloric acid or phosphoric acid, or food gradealkali, usually sodium hydroxide as required.

The diluted and optionally pH adjusted soy protein solution has aconductivity of generally below about 95 mS, preferably about 4 to about36 mS.

The aqueous soy protein solution may be subjected to a heat treatment toinactivate heat labile anti-nutritional factors, such as trypsininhibitors, present in such solution as a result of extraction from thesoy protein source material during the extraction step. Such a heatingstep also provides the additional benefit of reducing the microbialload. Generally, the protein solution is heated to a temperature ofabout 70° to about 160° C., preferably about 80° to about 120° C., morepreferably about 85° C. to about 95° C. for about 10 seconds to about 60minutes, preferably about 30 seconds to about 5 minutes. The heattreated soy protein solution then may be cooled for further processingas described below, to a temperature of about 2° C. to about 65° C.,preferably about 20° to about 35° C.

The optionally diluted, optionally pH adjusted and optionally heattreated protein solution may optionally be polished by any convenientmeans, such as by filtering to remove any residual particulates.

The resulting aqueous soy protein solution may be directly dried toproduce a soy protein product. In order to provide a soy protein producthaving a decreased impurities content and a reduced salt content, suchas a soy protein isolate, the aqueous soy protein solution may beprocessed prior to drying.

The aqueous soy protein solution may be concentrated to increase theprotein concentration thereof while maintaining the ionic strengththereof substantially constant. Such concentration generally is effectedto provide a concentrated soy protein solution having a proteinconcentration of about 50 to about 300 g/L, preferably about 100 toabout 200 g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the foodgrade salt but also low molecular weight materials extracted from thesource material, such as carbohydrates, pigments, low molecular weightproteins and anti-nutritional factors, such as trypsin inhibitor, whichthemselves are low molecular weight proteins. The molecular weightcut-off of the membrane is usually chosen to ensure retention of asignificant proportion of the protein in the solution, while permittingcontaminants to pass through having regard to the different membranematerials and configurations.

The concentrated soy protein solution then may be subjected to adiafiltration step using water or a dilute saline solution. Thediafiltration solution may be at its natural pH or at a pH equal to thatof the protein solution being diafiltered or at any pH value in between.Such diafiltration may be effected using from about 2 to about 40volumes of diafiltration solution, preferably about 5 to about 25volumes of diafiltration solution. In the diafiltration operation,further quantities of contaminants are removed from the aqueous soyprotein solution by passage through the membrane with the permeate. Thispurifies the aqueous protein solution and may also reduce its viscosity.The diafiltration operation may be effected until no significant furtherquantities of contaminants or visible colour are present in the permeateor until the retentate has been sufficiently purified so as, when dried,to provide a soy protein isolate with a protein content of at leastabout 90 wt % (N×6.25) d.b. Such diafiltration may be effected using thesame membrane as for the concentration step. However, if desired, thediafiltration step may be effected using a separate membrane with adifferent molecular weight cut-off, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 1,000,000Daltons, preferably about 5,000 to about 100,000 Daltons, having regardto different membrane materials and configuration.

Alternatively, the diafiltration step may be applied to the aqueousprotein solution prior to concentration or to the partially concentratedaqueous protein solution. Diafiltration may also be applied at multiplepoints during the concentration process. When diafiltration is appliedprior to concentration or to the partially concentrated solution, theresulting diafiltered solution may then be additionally concentrated.The viscosity reduction achieved by diafiltering multiple times as theprotein solution is concentrated may allow a higher final, fullyconcentrated protein concentration to be achieved. This reduces thevolume of material to be dried.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recoveredcontains less than about 90 wt % protein (N×6.25) d.b., such as at leastabout 60 wt % protein (N×6.25) d.b. By partially concentrating and/orpartially diafiltering the aqueous soy protein solution, it is possibleto only partially remove contaminants. This protein solution may then bedried to provide a soy protein product with lower levels of purity. Thesoy protein product is still able to produce clear protein solutionsunder acidic conditions.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The concentration step and the optional diafiltration step may beeffected at any convenient temperature, generally about 2° C. to about65° C., preferably about 20° C. to about 35° C., and for the period oftime to effect the desired degree of concentration and diafiltration.The temperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

There are two main trypsin inhibitors in soy, namely the Kunitzinhibitor, which is a heat-labile molecule with a molecular weight ofapproximately 21,000 Daltons, and the Bowman-Birk inhibitor, a moreheat-stable molecule with a molecular weight of about 8,000 Daltons. Thelevel of trypsin inhibitor activity in the final soy protein product canbe controlled by manipulation of various process variables.

As noted above, heat treatment of the aqueous soy protein solution maybe used to inactivate heat-labile trypsin inhibitors. The partiallyconcentrated or fully concentrated soy protein solution may also be heattreated to inactivate heat labile trypsin inhibitors. When the heattreatment is applied to the partially concentrated soy protein solution,the resulting heat treated solution may then be additionallyconcentrated.

In addition, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size (such asabout 30,000 to about 1,000,000 Da), operating the membrane at elevatedtemperatures (such as about 30° C. to about 65° C.) and employinggreater volumes of diafiltration medium (such as about 20 to about 40volumes).

Extracting and/or membrane processing the protein solution at a lower pH(1.5-3.0) may reduce the trypsin inhibitor activity relative toprocessing the solution at higher pH (3.0-5.0). When the proteinsolution is concentrated and diafiltered at the low end of the pH range,it may be desired to raise the pH of the retentate prior to drying. ThepH of the concentrated and diafiltered protein solution may be raised tothe desired value, for example pH 3, by the addition of any convenientfood grade alkali such as sodium hydroxide. If it is desired to lowerthe pH of the retentate prior to drying, this may be done so by theaddition of any convenient food grade acid such as hydrochloric acid orphosphoric acid.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the soyprotein source material in the extraction step, may be added to theclarified aqueous soy protein solution following removal of residual soyprotein source material, may be added to the concentrated proteinsolution before or after diafiltration or may be dry blended with thedried soy protein product. The addition of the reducing agent may becombined with a heat treatment step and the membrane processing steps,as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by eliminating or reducing theintensity of the heat treatment step, not utilizing reducing agents,operating the concentration and diafiltration steps at the higher end ofthe pH range (3.0 to 5.0), utilizing a concentration and diafiltrationmembrane with a smaller pore size, operating the membrane at lowertemperatures and employing fewer volumes of diafiltration medium.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076. Alternatively, defatting of theconcentrated and optionally diafiltered protein solution may be achievedby any other convenient procedure.

The concentrated and optionally diafiltered aqueous protein solution maybe treated with an adsorbent, such as powdered activated carbon orgranulated activated carbon, to remove colour and/or odour compounds.Such adsorbent treatment may be carried out under any convenientconditions, generally at the ambient temperature of the concentratedprotein solution. For powdered activated carbon, an amount of about0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v, isemployed. The adsorbent may be removed from the soy protein solution byany convenient means, such as by filtration.

The concentrated and optionally diafiltered soy protein solutionresulting from the optional defatting and optional adsorbent treatmentstep may be subjected to a pasteurization step to reduce the microbialload. Such pasteurization may be effected under any desiredpasteurization conditions. Generally, the concentrated and optionallydiafiltered soy protein solution is heated to a temperature of about 55°to about 70° C., preferably about 60° to about 65° C., for about 30seconds to about 60 minutes, preferably about 10 minutes to about 15minutes. The pasteurized concentrated and diafiltered soy proteinsolution then may be cooled for drying or further processing, preferablyto a temperature of about 20° to about 35° C.

In accordance with one aspect of the current invention, the concentratedand optionally diafiltered soy protein solution may be dried by anyconvenient technique, such as spray drying or freeze drying, to yieldthe soy protein product. The dry soy protein product has a proteincontent in excess of about 60 wt % (N×6.25) d.b. Preferably, the dry soyprotein product is an isolate with a high protein content, in excess ofabout 90 wt % protein, preferably at least about 100 wt % (N×6.25) d.b.

In another aspect of the invention, the concentrated protein solutionresulting from the concentration step and optional diafiltration step,optional defatting step, optional adsorbent treatment step and optionalpasteurization step, is optionally adjusted in pH within the range ofabout 1.5 to about 7.0, preferably to about 4.0 to about 7.0, morepreferably to about 5.0 to about 7.0 and then diluted by mixing theconcentrated protein solution with water having the volume required toachieve the degree of dilution desired. When the intent is to separateprecipitated protein from the residual aqueous phase, termed thesupernatant, as is the case for this aspect of the current invention,the degree of dilution is generally about 5 fold to about 25 fold,preferably about 10 fold to about 20 fold. The water with which theconcentrated protein solution is mixed preferably has a temperature ofabout 1° to about 65° C., preferably about 20° to about 35° C.

In a batch operation, the batch of concentrated protein solution isadded to a static body of water having the desired volume, as discussedabove. Dilution of the concentrated protein solution decreases the ionicstrength and causes the formation of the protein precipitate. In thebatch procedure, the protein precipitate is allowed to settle in thebody of water. The settling may be assisted, such as by centrifugation.Such induced settling decreases the moisture content and the occludedsalt content of the precipitated protein.

Alternatively, the dilution operation may be carried out continuously bycontinuously passing the concentrated protein solution to one inlet of aT-shaped pipe, while the diluting water is fed to the other inlet of theT-shaped pipe, permitting mixing in the pipe. The diluting water is fedinto the T-shaped pipe at a rate sufficient to achieve the desireddegree of dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and the diluting waterin the pipe initiates the formation of protein precipitate and themixture is continuously fed from the outlet of the T-shaped pipe into asettling vessel, from which, when full, supernatant is permitted tooverflow. The mixture preferably is fed into the body of liquid in thesettling vessel in a manner which minimizes turbulence within the bodyof liquid.

In the continuous procedure, the protein precipitate is allowed tosettle in the settling vessel and the procedure is continued until adesired quantity of the precipitate has accumulated in the bottom of thesettling vessel, whereupon the accumulated precipitate is removed fromthe settling vessel. In lieu of settling by sedimentation, theprecipitate may be separated continuously by centrifugation.

By the utilization of a continuous process for the recovery of soyprotein precipitate as compared to the batch process, the initialprotein extraction step can be significantly reduced in time for thesame level of protein extraction. In addition, in a continuousoperation, there is less chance of contamination than in a batchprocedure, leading to higher product quality and the process can becarried out in more compact equipment.

Settled precipitate is separated from the residual aqueous phase orsupernatant, such as by decantation of the residual aqueous phase fromthe settled mass or by centrifugation. The precipitate may be washed toremove residual supernatant, such as with about 1 to about 10,preferably about 2 to about 3 volumes of water and then the precipitaterecovered again, as above. The optionally washed precipitate may be usedin the wet form or may be dried, by any convenient technique, such asspray drying or freeze drying, to a dry form. The dry precipitate has ahigh protein content, in excess of about 60 wt % protein, preferably atleast about 90 wt % protein (N×6.25), and more preferably at least about100 wt % (N×6.25).

The supernatant arising from the dilution step may be dried to provide asoy protein product. Alternatively, the supernatant may be processed todecrease the impurity content thereof and/or the trypsin inhibitoractivity thereof, by any convenient means such as pH adjustment and/orheat treatment and/or membrane processing. The processed supernatant maythen be dried to provide a soy protein product.

As mentioned above, settled protein precipitate formed in the dilutionstep may be directly dried to yield the protein product. Alternatively,the wet protein precipitate may be re-suspended in water, such as about2 to about 3 volumes, and re-solubilized by adjusting the pH of thesample to about 1.5 to about 4.4, preferably about 2.0 to about 4.0,using any convenient acid, such as hydrochloric acid or phosphoric acid.The re-solubilized protein solution then may be dried by any convenienttechnique, such as spray drying or freeze drying to a dry form. The dryprotein product has a protein content in excess of about 60 wt %protein, preferably at least about 90 wt % protein, more preferably atleast about 100 wt % protein (N×6.25).

As a further alternative, the re-solubilized soy protein solution may besubjected to a heat treatment to inactivate any remaining heat labileanti-nutritional factors. Such a heating step also provides theadditional benefit of reducing the microbial load. Generally, theprotein solution is heated to a temperature of about 70° to about 160°C., preferably about 80° to about 120° C., more preferably about 85° toabout 95° C., for about 10 seconds to about 60 minutes, preferably about30 seconds to about 5 minutes. The heat treated soy protein solutionthen may be cooled for further processing as described below, to atemperature of about 2° to about 65° C., preferably about 20° to about35° C.

The re-solubilized and optionally heat treated protein solution mayoptionally be polished by any convenient means, such as by filtering, toremove any residual particulates.

The re-solubilized, optionally heat treated, optionally polished clearprotein solution, may be concentrated to increase the proteinconcentration thereof. Such concentration is effected using anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes with a suitable molecular weight cut-offpermitting low molecular weight species, including salt, carbohydrates,pigments, trypsin inhibitors and other low molecular weight materialsextracted from the protein source material, to pass through themembrane, while retaining a significant proportion of the soy protein inthe solution. Ultrafiltration membranes having a molecular weightcut-off of about 3,000 to 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfiguration, may be used. Concentration of the protein solution inthis way also reduces the volume of liquid required to be dried torecover the protein. The protein solution generally is concentrated to aprotein concentration of about 50 g/L to about 300 g/L, preferably about100 to about 200 g/L, prior to drying. Such concentration operation maybe carried out in a batch mode or in a continuous operation, asdescribed above.

The soy protein solution may be subjected to a diafiltration step beforeor after complete concentration using water. The water may be at itsnatural pH or at a pH equal to that of the protein solution beingdiafiltered or at any pH value in between. Such diafiltration may beeffected using from about 2 to about 40 volumes of diafiltrationsolution, preferably about 5 to about 25 volumes of diafiltrationsolution. In the diafiltration operation, further quantities ofcontaminants are removed from the clear aqueous soy protein solution bypassage through the membrane with the permeate. The diafiltrationoperation may be effected until no significant further quantities ofcontaminants or visible colour are present in the permeate or until theretentate has been sufficiently purified so as, when dried, to provide asoy protein product with the desired protein content, preferably anisolate with a protein content of at least about 90 wt % (N×6.25) d.b.Such diafiltration may be effected using the same membrane as for theconcentration step. However, if desired, the diafiltration step may beeffected using a separate membrane with a different molecular weightcut-off, such as a membrane having a molecular weight cut-off in therange of about 3,000 to about 1,000,000 Daltons, preferably about 5,000to about 100,000 Daltons, having regard to different membrane materialsand configuration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6.25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity. The soyprotein product is still able to produce clear protein solutions underacidic conditions.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The optional concentration step and the optional diafiltration step maybe effected at any convenient temperature, generally about 2° to about65° C., preferably about 20° to about 35° C., and for the period of timeto effect the desired degree of concentration and diafiltration. Thetemperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

As previously noted, heat treatment of the re-solubilized aqueous soyprotein solution may be used to inactivate remaining heat-labile trypsininhibitors. Partially concentrated or fully concentrated re-solubilizedsoy protein solution may also be heat treated to inactivate heat labiletrypsin inhibitors.

In addition, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such as30,000 to 1,000,000 Daltons, operating the membrane at elevatedtemperatures, such as 30° to 65° C. and employing greater volumes ofdiafiltration medium, such as 20 to 40 volumes.

Membrane processing the protein solution at a lower pH (1.5 to 3) mayreduce the trypsin inhibitor activity relative to processing thesolution at higher pH (3 to 4.4). When the protein solution isconcentrated and diafiltered at the low end of the pH range, it may bedesired to raise the pH of the retentate prior to drying. The pH of theconcentrated and diafiltered protein solution may be raised to thedesired value, for example pH 3, by the addition of any convenient foodgrade alkali such as sodium hydroxide.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added to the wetprotein precipitate resulting from the dilution step, may be added tothe protein solution formed by re-solubilizing the precipitate, may beadded to the concentrated solution before or after diafiltration or maybe dry blended with the dried soy protein product. The addition of thereducing agent may be combined with a heat treatment step and themembrane processing steps, as described above.

If it is desired to retain remaining active trypsin inhibitors in theconcentrated protein solution, this can be achieved by eliminating orreducing the intensity of the heat treatment step, not utilizingreducing agents, operating the concentration and diafiltration steps atthe higher end of the pH range (3 to 4.4), utilizing a concentration anddiafiltration membrane with a smaller pore size, operating the membraneat lower temperatures and employing fewer volumes of diafiltrationmedium.

The re-solubilized, optionally concentrated and optionally diafilteredaqueous protein solution may be treated with an adsorbent, such aspowdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the protein solution. For powdered activated carbon, anamount of about 0.025% to about 5% w/v, preferably about 0.05% to about2% w/v, is employed. The adsorbent may be removed from the soy proteinsolution by any convenient means, such as by filtration.

The re-solubilized, optionally concentrated and optionally diafilteredaqueous soy protein solution then may be dried by any convenienttechnique, such as spray drying or freeze drying. The dry soy proteinproduct has a protein content of at least about 60 wt % (N×6.25) d.b.,preferably in excess of about 90 wt % (N×6.25) d.b., more preferably atleast about 100 wt % (N×6.25) d.b.

In accordance with another aspect of the current invention, the mixtureof concentrated protein solution and dilution water may be processedwithout a fractionation step. In such a case, the degree of dilution isgenerally about 1 to 25 fold, preferably about 3 to about 12 fold. Thewater with which the concentrated protein solution is mixed has atemperature of about 1° to about 65° C., preferably about 20° C. toabout 35° C.

The dilution water, containing the deposited protein precipitate, isadjusted in pH to about 1.5 to about 4.4, preferably about 2.0 to about4.0, using any convenient acid, such as hydrochloric acid or phosphoricacid. The adjustment in pH causes the resolubilization of proteindeposited by dilution. The protein solution may be used in the wet formor may be dried, by any convenient technique, such as spray drying orfreeze drying, to a dry form.

As a further alternative, the protein solution formed by pH adjustingthe mixture of protein precipitate and supernatant may be processedutilizing the same steps as described above for the isolated precipitateresolubilized by pH adjustment.

The optionally concentrated, optionally diafiltered, optionally heattreated, optionally polished, optional adsorbent treated aqueous soyprotein solution then may be dried by any convenient technique, such asspray drying or freeze drying. The dry soy protein product has a proteincontent in excess of about 60 wt % protein, preferably at least about 90wt %, more preferably about 100 wt % (N×6.25) d.b.

The soy protein products produced herein are soluble in an acidicaqueous environment, making the product ideal for incorporation intobeverages, both carbonated and uncarbonated, to provide proteinfortification thereto. Such beverages have a wide range of acidic pHvalues, ranging from about 2.5 to about 5. The soy protein productsprovided herein may be added to such beverages in any convenientquantity to provide protein fortification to such beverages, forexample, at least about 5 g of the soy protein per serving. The addedsoy protein product dissolves in the beverage and does not impair theclarity of the beverage, even after thermal processing. The soy proteinproduct may be blended with dried beverage prior to reconstitution ofthe beverage by dissolution in water. In some cases, modification of thenormal formulation of the beverages to tolerate the composition of theinvention may be necessary where components present in the beverage mayadversely affect the ability of the composition to remain dissolved inthe beverage.

EXAMPLES Example 1

This Example illustrates the preparation of transparent, heat stableprotein solutions utilizing extraction with calcium chloride solution atlow pH.

Soy white flakes (10 g) were combined with 0.15M calcium chloridesolution (100 ml) and the pH of the samples adjusted immediately to 4.8and 1.5 with HCl. The samples were extracted at room temperature for 30minutes using a magnetic stirrer. The pH of the samples was monitoredand adjusted two times during the 30 minute extraction. The extract wasseparated from the spent meal by centrifugation at 10,200 g for 10minutes and the centrates further clarified by filtration using 25 μmpore size filter paper. The clarity of the filtrates was measured usinga HunterLab ColorQuest XE operated in transmission mode to supply apercentage haze reading. The samples were then diluted with one volumeof reverse osmosis purified water and the haze level measured again. ThepH of the diluted samples was then adjusted to 3 using either HCl orNaOH as necessary. The haze level of the pH adjusted samples was thenanalyzed. The samples were then heat treated to 95° C. for 30 seconds,immediately cooled to room temperature in ice water and the haze levelre-assessed.

The haze values determined for the various samples are shown in Tables 1and 2.

TABLE 1 Haze values for the treatment of samples from extraction withcalcium chloride solution at pH 1.5 sample haze (%) filtrate 27.8diluted filtrate 17.1 diluted filtrate at pH 3 16.8 diluted filtrate atpH 3 after heat treatment 10.4

TABLE 2 Haze values for the treatment of samples from extraction withcalcium chloride solution at pH 4.8 sample haze (%) filtrate 36.2diluted filtrate 99.1 diluted filtrate at pH 3 8.4 diluted filtrate atpH 3 after heat treatment 6.0

As may be seen from the results presented in Tables 1 and 2, the initialfiltrates were somewhat hazy, however improved clarity may have beenobtained by utilizing a finer filter. Dilution with one volume of waterimproved the clarity of the pH 1.5 sample but introduced precipitationin the pH 4.8 sample. Adjusting the pH of the diluted samples to 3 gavegood clarity to the sample that was originally at pH 4.8, while thesample that was originally at pH 1.5 had perhaps a slight haze. Afterheat treatment both samples were considered clear.

Example 2

This Example illustrates the preparation of a soy protein isolate inaccordance with one embodiment of the invention.

20 kg of defatted, minimally heat treated soy flour was added to 200 Lof 0.15 M calcium chloride solution at ambient temperature and agitatedfor 30 minutes to provide an aqueous protein solution. Immediately afterthe flour was dispersed in the calcium chloride solution, the pH of thesystem was adjusted to 3 by the addition of diluted HCl. The pH wasmonitored and corrected to 3 periodically over the course of the 30minute extraction. The residual soy flour was removed by centrifugationto yield 174 L of protein solution having a protein content of 3.37% byweight. The protein solution was then combined with 174 L of reverseosmosis purified water and the pH corrected to 3. This solution was thenpolished by filtration to yield 385 L of filtered protein solutionhaving a protein content of 1.21% by weight.

The filtered protein solution was reduced in volume to 25 L byconcentration on a PVDF membrane having a molecular weight cutoff of5,000 Daltons. The concentrated protein solution was then diafilteredwith 125 L of reverse osmosis purified water. The resulting diafiltered,concentrated protein solution had a protein content of 14.51% by weightand represented a yield of 81.3 wt % of the filtered protein solution.The diafiltered, concentrated protein solution was then dried to yield aproduct found to have a protein content of 99.18% (N×6.25) d.b. Theproduct was termed S005-A13-09A S703.

Sufficient S005-A13-09A S703 to supply 0.48 g of protein was dissolvedin 15 ml reverse osmosis purified water and the solution colour andclarity assessed using a HunterLab Color Quest XE instrument operated intransmission mode. The pH of the solution was measured with a pH meter.

The pH, colour and clarity values are set forth in the following Table3:

TABLE 3 pH and HunterLab scores for solution of S005-A13-09A S703 samplepH L* a* b* haze (%) S703 3.12 87.31 0.67 18.99 43.9

As may be seen from Table 3, the solution of S703 in water wassemi-transparent, not transparent. The relatively high level of haze inthis sample resulted in the L* value being somewhat lower than expected.

The colour of the dry powder was also assessed with the HunterLab ColorQuest XE instrument in reflectance mode. The colour values are set forthin the following Table 4:

TABLE 4 HunterLab scores for S005-A13-09A S703 dry powder sample L* a*b* S703 85.67 0.05 10.57

As may be seen from Table 4, the dry product was very light in colour.

Example 3

This Example contains an evaluation of the heat stability in water ofthe soy protein isolate produced by the method of Example 2 (S703).

A solution of S005-A13-09A S703 was prepared by dissolving sufficientprotein powder to supply 0.8 g protein in 40 ml RO water then the pHadjusted to 3. The clarity of this solution was assessed by hazemeasurement with the HunterLab Color Quest XE instrument. The solutionwas then heated to 95° C., held at this temperature for 30 seconds andthen immediately cooled to room temperature in an ice bath. The clarityof the heat treated solution was then measured again.

The clarity of the protein solution before and after heating is setforth in the following Table 5:

TABLE 5 Effect of heat treatment on clarity of S005-A13-09A S703solution sample haze (%) before heating 43.6 after heating 30.7

As can be seen from the results in Table 5, it was found that theinitial solution of S005-A13-09A S703 was quite hazy. However, thesolution was heat stable, with the haze level actually reduced somewhatby the heat treatment.

Example 4

This Example contains an evaluation of the solubility in water of thesoy protein isolate produced by the method of Example 2 (S703).Solubility was tested based on protein solubility (termed proteinmethod, a modified version of the procedure of Morr et al., J. Food Sci.50:1715-1718) and total product solubility (termed pellet method).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared atnatural pH. For the pH adjusted samples, the pH was measured andcorrected two times during the 60 minutes stirring. After the 60 minutesof stirring, the samples were made up to 50 ml total volume with ROwater, yielding a 1% w/v protein dispersion. The protein content of thedispersions was measured using a Leco FP528 Nitrogen Determinator.Aliquots (20 ml) of the dispersions were then transferred to pre-weighedcentrifuge tubes that had been dried overnight in a 100° C. oven thencooled in a desiccator and the tubes capped. The samples werecentrifuged at 7,800 g for 10 minutes, which sedimented insolublematerial and yielded a clear supernatant. The protein content of thesupernatant was measured by Leco analysis and then the supernatant andthe tube lids were discarded and the pellet material dried overnight inan oven set at 100° C. The next morning the tubes were transferred to adesiccator and allowed to cool. The weight of dry pellet material wasrecorded. The dry weight of the initial protein powder was calculated bymultiplying the weight of powder used by a factor of ((100−moisturecontent of the powder (%))/100). Solubility of the product was thencalculated two different ways:Solubility(protein method)(%)=(% protein in supematant/% protein ininitial dispersion)×100  1)Solubility(pellet method)(%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100  2)

The natural pH value of the protein isolate produced in Example 1 inwater (1% protein) is shown in Table 6:

TABLE 6 Natural pH of S703 solution prepared in water at 1% proteinBatch Product Natural pH S005-A13-09A S703 3.36

The solubility results obtained are set forth in the following Tables 7and 8:

TABLE 7 Solubility of S703 at different pH values based on proteinmethod Solubility (protein method) (%) Batch Product pH 2 pH 3 pH 4 pH 5pH 6 pH 7 Nat. pH S005- S703 95.8 100 81.7 0.0 71.7 100 100 A13-09A

TABLE 8 Solubility of S703 at different pH values based on pellet methodSolubility (pellet method) (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH7 Nat. pH S005- S703 95.9 95.9 83.8 11.9 69.2 96.0 95.2 A13-09A

As can be seen from the results of Tables 7 and 8, the S703 product washighly soluble at pH values 2, 3 and 7 as well as at the natural pH. Thesolubility was slightly lower at pH 4.

Example 5

This Example contains an evaluation of the clarity in water of the soyprotein isolate produced by the method of Example 2 (S703).

The clarity of the 1% w/v protein solutions prepared as described inExample 4 was assessed by measuring the absorbance at 600 nm, with alower absorbance score indicating greater clarity. Analysis of thesamples on a HunterLab ColorQuest XE instrument in transmission modealso provided a percentage haze reading, another measure of clarity.

The clarity results are set forth in the following Tables 9 and 10:

TABLE 9 Clarity of S703 solution at different pH values as assessed byA600 A600 Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH S005- S7030.098 0.152 1.381 >3.0 1.876 0.155 0.192 A13-09A

TABLE 10 Clarity of S703 solution at different pH values as assessed byHunterLab analysis HunterLab haze reading (%) Batch Product pH 2 pH 3 pH4 pH 5 pH 6 pH 7 Nat. pH S005- S703 12.0 20.8 86.3 91.6 90.0 19.7 29.8A13-09A

As can be seen from the results of Tables 9 and 10, the solutions ofS703 were clear to slightly hazy at pH 2-3. A slightly hazy solution wasalso obtained at pH 7.

Example 6

This Example contains an evaluation of the solubility in a soft drink(Sprite) and sports drink (Orange Gatorade) of the soy protein isolateproduced by the method of Example 2 (S703). The solubility wasdetermined with the protein added to the beverages with no pH correctionand again with the pH of the protein fortified beverages adjusted to thelevel of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply 1 g of protein was weighed into abeaker and a small amount of beverage was added and stirred until asmooth paste formed. Additional beverage was added to bring the volumeto 50 ml, and then the solutions were stirred slowly on a magneticstirrer for 60 minutes to yield a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a Leco FP528 NitrogenDeterminator then an aliquot of the protein containing beverages wascentrifuged at 7,800 g for 10 minutes and the protein content of thesupernatant measured.Solubility(%)=(% protein in supernatant/% protein in initialdispersion)×100

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) (3.39) and sports drink (Orange Gatorade) (3.19) withoutprotein was measured. A sufficient amount of protein powder to supply 1g of protein was weighed into a beaker and a small amount of beveragewas added and stirred until a smooth paste formed. Additional beveragewas added to bring the volume to approximately 45 ml, and then thesolutions were stirred slowly on a magnetic stirrer for 60 minutes. ThepH of the protein containing beverages was measured and then adjusted tothe original no-protein pH with HCl or NaOH as necessary. The totalvolume of each solution was then brought to 50 ml with additionalbeverage, yielding a 2% protein w/v dispersion. The protein content ofthe samples was analyzed using a Leco FP528 Nitrogen Determinator thenan aliquot of the protein containing beverages was centrifuged at 7,800g for 10 minutes and the protein content of the supernatant measured.Solubility(%)=(% protein in supernatant/% protein in initialdispersion)×100

The results obtained are set forth in the following Table 11:

TABLE 11 Solubility of S703 in Sprite and Orange Gatorade no pHcorrection pH correction Solubility Solubility Solubility (%) inSolubility (%) in (%) in Orange (%) in Orange Batch Product SpriteGatorade Sprite Gatorade S005-A13-09A S703 94.8 100 99.0 93.6

As can be seen from the results of Table 11, the S703 was highly solublein the Sprite and the Orange Gatorade. As S703 is an acidified product,protein addition had little effect on beverage pH.

Example 7

This Example contains an evaluation of the clarity in a soft drink andsports drink of the soy protein isolate produced by the method ofExample 2 (S703).

The clarity of the 2% w/v protein dispersions prepared in soft drink(Sprite) and sports drink (Orange Gatorade) in Example 6 were assessedusing the methods described in Example 5. For the absorbancemeasurements at 600 nm, the spectrophotometer was blanked with theappropriate beverage before the measurement was performed.

The results obtained are set forth in the following Tables 12 and 13:

TABLE 12 Clarity (A600) of S703 in Sprite and Orange Gatorade no pHcorrection pH correction A600 in A600 in A600 in Orange A600 in OrangeBatch Product Sprite Gatorade Sprite Gatorade S005-A13-09A S703 0.4600.404 0.471 0.539

TABLE 13 HunterLab haze readings for S703 in Sprite and Orange Gatoradeno pH correction pH correction haze haze haze (%) in haze (%) in (%) inOrange (%) in Orange Batch Product Sprite Gatorade Sprite Gatorade noprotein 0.0 44.0 0.0 44.0 S005-A13-09A S703 58.5 74.3 55.6 79.5

As can be seen from the results of Tables 12 and 13, the good solubilityresults obtained for the S703 in the Sprite and the Orange Gatorade didnot translate to clarity in these beverages. In fact, the resultingsolutions were quite hazy.

Example 8

This Example illustrates the preparation of soy protein isolates inaccordance with other embodiments of the invention.

100 g of defatted soy white flake was added to 1000 ml of 0.15 M CaCl₂solution at ambient temperature and agitated for 30 minutes to providean aqueous protein solution. Immediately after the flake was wetted withthe calcium chloride solution, the pH of the system was adjusted to 4.5with a solution of hydrochloric acid. The pH was monitored and correctedperiodically throughout the 30 minute extraction. After the extractionstep, the residual soy white flake was removed and the resulting proteinsolution was clarified by centrifugation and filtration to produce 578ml of filtered protein solution having a protein content of 2.05% byweight.

530 ml of the protein extract solution was reduced to 45 ml on apolyethersulfone membrane having a molecular weight cutoff of 10,000Daltons, producing a concentrated protein solution with a proteincontent of 19.40% by weight. The concentrated protein solution was thendivided into two portions.

20 ml of the concentrated protein solution at 24° C. was diluted into200 ml of reverse osmosis (RO) purified water having a temperature of24° C. A white cloud formed and was allowed to settle. The sample thenwas centrifuged to separate the protein precipitate from the supernatantfraction. 5.72 g of wet protein precipitate was collected thenresolublized in 20 ml of RO water with HCl solution added to reduce thepH to 2.99. The resolubilized protein precipitate, recovered in a yieldof 23.8 wt % of the filtered protein solution, was freeze dried toprovide a product given the designation S703-7300. The dried product wasfound to have a protein content of 101.75% (N×6.25) d.b.

Another 21 ml of the concentrated protein solution at 24° C. was dilutedinto 210 ml of RO water having a temperature of 24° C. The pH of thesample was then lowered from 4.76 to 2.98 with HCl solution. 220 ml ofthe acidified solution was reduced in volume to 33 ml on apolyethersulfone membrane having a molecular weight cutoff of 10,000Daltons, producing a concentrated protein solution with a proteincontent of 9.76% by weight. This concentrated protein solution,recovered in a yield of 30.1 wt % of the filtered protein solution, wasfreeze dried to provide a product given the designation S703-7301. Thedried product was found to have a protein content of 92.21% (N×6.25)d.b.

Solutions of S703-7300 and S703-7301 were prepared by dissolvingsufficient powder to supply 0.48 g protein in 15 ml of RO water. Thecolour and clarity of the solutions were assessed using a HunterLabColorQuest XE operated in transmission mode. The pH of the solutions wasmeasured with a pH meter.

The pH, colour and clarity values are set forth in the following Table14.

TABLE 14 pH and HunterLab scores for S703-7300 and S703-7301 solutionssample pH L* a* b* haze (%) S703-7300 2.83 88.67 0.71 15.57 38.9S703-7301 3.10 88.71 0.80 14.84 30.8

As may be seen from the results presented in Table 14, the solutions ofS703-7300 and S703-7301 were translucent and light in colour.

Example 9

This Example illustrates the generation of a protein precipitate upondilution of concentrated protein solutions prepared at low pH thenadjusted in pH prior to the dilution step.

100 g of defatted soy white flake was added to 1000 ml of 0.15 M CaCl₂solution at ambient temperature and agitated for 30 minutes to providean aqueous protein solution. Immediately after the flake was wetted withthe calcium chloride solution, the pH of the system was adjusted to 3.0with a solution of hydrochloric acid. The pH was monitored and correctedperiodically throughout the 30 minute extraction. After the extractionstep, the residual soy white flake was removed and the resulting proteinsolution was clarified by centrifugation and filtration to produce 568ml of filtered protein solution having a protein content of 2.78% byweight.

550 ml of the protein extract solution was reduced to 84 ml on apolyethersulfone membrane having a molecular weight cutoff of 10,000Daltons, producing a concentrated protein solution with a proteincontent of 15.18% by weight.

The ultrafiltration retentate, having a pH of 3.11 was divided intoaliquots and the pH adjusted with 6M NaOH and 0.5M HCl as necessary toapproximately 4, 5, 6 or 7. The protein content of the pH adjustedretentate samples was measured. Aliquots of the pH adjusted retentatesamples were clarified by centrifugation at 7,800 g for 10 minutes thenthe protein content of the centrates determined. Additional aliquots ofthe pH adjusted retentate samples were diluted with 10 volumes of ROwater, mixed with a vortex and the pH, conductivity, A600 and proteincontent of the diluted samples determined. The diluted samples wereclarified by centrifugation at 7,800 g for 10 minutes then the proteincontent of the centrate was determined.

Raising the pH of the retentate caused all the samples to becomecloudier, regardless of the final pH. Determination of the proteincontent before and after clarification indicated that about 20% of theprotein in the sample was precipitated by the pH adjustment. (Table 15).

TABLE 15 Protein content of pH adjusted retentate samples before andafter clarification % w/w % w/w Retentate A600 protein protein % ofprotein adjusted to before before after precipitated by pH clarificationclarification clarification pH adjustment 3.11 0.437 15.18 15.54 0.004.00 2.667 15.13 12.06 20.3 5.01 2.879 14.94 11.75 21.4 6.04 2.877 15.0211.99 20.2 7.00 2.889 14.91 12.03 19.3

Dilution of the pH adjusted retentate samples resulted in samples thatwere very cloudy, particularly when the retentate was at pH 4 and higher(Table 16). Analysis of the protein concentration of the samples, beforeand after clarification indicated that some protein was precipitated atall pH values, but particularly when the retentate pH was 4 or greaterbefore the dilution step. The high degree of protein precipitation inthe pH 4-7 samples indicates that the dilution step is introducingprotein precipitation beyond that induced by the pH adjustment.

TABLE 16 Properties of pH adjusted (no clarification) retentate samplesafter dilution % w/w % w/w % of Retentate protein protein proteinadjusted Cond (no clari- after clari- precipitated to pH pH (mS) A600fication) fication by dilution 3.11 3.34 3.27 1.625 1.33 0.89 33.1 4.004.36 3.02 2.601 1.01 0.08 92.1 5.01 5.25 2.82 2.425 0.96 0.02 97.9 6.046.24 2.96 2.574 0.99 0.12 87.9 7.00 7.03 2.90 2.706 1.13 0.13 88.5

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a methodof producing a soy protein isolate which is soluble in acid media, basedon extraction of a soy protein source material using aqueous calciumchloride solution at low pH. Modifications are possible within the scopeof this invention.

What we claim is:
 1. A method of producing a soy protein product havinga soy protein content of at least about 60 wt % (N×6.25) on a dry weightbasis, which comprises: (a) extracting a soy protein source with anaqueous calcium salt solution at a pH of about 1.5 to about 5 to causesolubilization of soy protein from the soy protein source and to form anaqueous soy protein solution, (b) at least partially separating theaqueous soy protein solution from residual soy protein source, and (c)following said separation step, diluting said aqueous soy proteinsolution to a conductivity of less than about 90 mS.
 2. The process ofclaim 1 wherein said extraction step is effected using an aqueouscalcium chloride solution having a concentration of less than about 1.0M.
 3. The process of claim 2 wherein said aqueous calcium chloridesolution has a concentration of about 0.10 to about 0.15 M.
 4. Theprocess of claim 1 wherein said extraction step is effected at atemperature of about 15° to about 35° C.
 5. The process of claim 1wherein said aqueous soy protein solution has a protein concentration ofabout 5 to about 50 g/L.
 6. The process of claim 5 wherein said proteinconcentration is about 10 to about 50 g/L.
 7. The process of claim 1wherein said aqueous calcium salt solution contains an antioxidant. 8.The process of claim 1 wherein, following said separation step, theseparated aqueous soy protein solution is treated with an adsorbent toremove colour and/or odour compounds from the separated aqueous soyprotein solution, prior to the dilution step.
 9. The process of claim 1wherein following said dilution step, the pH of the aqueous proteinsolution, is adjusted to a different value within the range of about 1.5to about 5.0.
 10. The process of claim 9 wherein the pH value isadjusted to about 1.5 to about 4.4.
 11. The process of claim 10 whereinthe pH value is adjusted to about 2.0 to 4.0.
 12. The process of claim 1wherein said aqueous soy protein solution is diluted with about 0.5 toabout 10 volumes of aqueous diluent such as water or dilute saltsolution to provide a conductivity of said soy protein solution of about4 to about 31 mS.
 13. The process of claim 1 wherein said diluent has atemperature of about 2° C. to about 70° C.
 14. The process of claim 13wherein said temperature is about 15° to about 65° C.
 15. The process ofclaim 14 wherein said temperature is about 20° to about 35° C.
 16. Theprocess of claim 9 wherein said soy protein solution, following thedilution and pH adjustment steps has a conductivity of less than about95 mS.
 17. The process of claim 16 wherein said conductivity is about 4to about 36 mS.
 18. The process of claim 1, wherein said aqueous proteinsolution is subjected to a heat treatment step to inactivate heat-labileanti-nutritional factors.
 19. The process of claim 18 wherein theanti-nutritional factors are heat-labile trypsin inhibitors.
 20. Theprocess of claim 18 wherein the heat treatment step also pasteurizes theaqueous soy protein solution.
 21. The process of claim 18, wherein saidheat treatment is effected at a temperature of about 70° to about 160°C. for about 10 seconds to about 60 minutes.
 22. The process of claim 21wherein said heat treatment is effected at a temperature of about 80° toabout 120° C. for about 10 seconds to about 5 minutes.
 23. The processof claim 22 wherein said heat treatment is effected at a temperature ofabout 85° C. to about 95° C. for about 30 seconds to about 5 minutes.24. The process of claim 18 wherein the heat treated soy proteinsolution is cooled to a temperature of about 2° to about 65° C. forfurther processing.
 25. The process of claim 24 wherein the heat treatedsoy protein solution is cooled to a temperature of about 20° to about35° C. for further processing.
 26. The process of claim 1 wherein thediluted soy protein solution is dried to provide a soy protein producthaving a soy protein content of at least about 60 wt % (N×6.25) d.b. 27.The process of claim 1 wherein the diluted soy protein solution isconcentrated while maintaining the ionic strength thereof substantiallyconstant to produce a concentrated soy protein solution having a proteinconcentration of about 50 to about 300 g/L and the concentrated soyprotein solution is optionally diafiltered.
 28. The process of claim 27wherein said concentrated soy protein solution has a proteinconcentration of about 100 to about 200 g/L.
 29. The process of claim 27wherein said concentration step is effected by ultrafiltration using amembrane having a molecular weight cut-off of about 3,000 to about1,000,000 Daltons.
 30. The process of claim 29 wherein said membrane hasa molecular weight cut-off of about 5,000 to about 100,000 Daltons. 31.The process of claim 27 wherein a diafiltration step is effected usingwater, dilute saline, acidified water or acidified dilute saline on thesoy protein solution before or after partial or complete concentrationthereof.
 32. The process of claim 31 wherein said diafiltration iseffected using about 2 to about 40 volumes of diafiltration solution.33. The process of claim 32 wherein said diafiltration is effected usingabout 5 to about 25 volumes of diafiltration solution.
 34. The processof claim 31 wherein said diafiltration is effected until no significantfurther quantities of contaminants or visible colour are present in thepermeate.
 35. The process of claim 31 wherein said diafiltration iseffected until the retentate has been sufficiently purified so as, whendried, to provide a soy protein isolate with a protein content of atleast about 90 wt % (N×6.25) d.b.
 36. The process of claim 31 whereinsaid diafiltration is effected using a membrane having a molecularweight cut-off of about 3,000 to about 1,000,000 Daltons.
 37. Theprocess of claim 36 wherein said membrane has a molecular weight cut-offof about 5,000 to about 100,000 Daltons.
 38. The process of claim 31wherein an antioxidant is present in the diafiltration medium during atleast part of the diafiltration step.
 39. The process of claim 27wherein said concentration step and optional diafiltration step arecarried out at a temperature of about 2° to about 65° C.
 40. The processof claim 39 wherein said temperature is about 20° to about 35° C. 41.The process of claim 27 wherein the concentrated and optionallydiafiltered soy protein solution is subjected to a heat treatment stepto inactivate heat-labile anti-nutritional factors, includingheat-labile trypsin inhibitors.
 42. The process of claim 41 wherein saidheat treatment is effected at a temperature of about 70° to about 160°C. for about 10 seconds to 60 minutes.
 43. The process of claim 42wherein the heat treated soy protein solution is cooled to a temperatureof about 2° to about 65° C. for further processing.
 44. The process ofclaim 1 wherein said soy protein solution is concentrated anddiafiltered to produce a concentrated and diafiltered soy proteinsolution which, when dried, provides a soy protein product having aprotein concentration of at least about 60 wt % (N×6.25) d.b.
 45. Theprocess of claim 27 wherein said concentrated and optionally diafilteredsoy protein solution is treated with an adsorbent to remove colourand/or odour compounds.
 46. The process of claim 27 wherein saidconcentrated and optionally diafiltered soy protein solution ispasteurized prior to drying.
 47. The process of claim 46 wherein saidpasteurization step is effected at a temperature of about 55° to about70° C. for about 30 seconds to about 60 minutes.
 48. The process ofclaim 47 wherein said pasteurization step is effected at a temperatureof about 60° to about 65° C. for about 10 to about 15 minutes.
 49. Theprocess of claim 35 wherein said concentrated and optionally diafilteredsoy protein solution is dried to provide a soy protein isolate having aprotein content of at least about 90 wt % (N×6.25) d.b.
 50. The processof claim 49 wherein said soy protein isolate has a protein content of atleast about 100 wt % (N×6.25) d.b.
 51. The process of claim 27 whereinthe concentration and/or optional diafiltration step are operated in amanner favourable to the removal of trypsin inhibitors.
 52. The processof claim 1 wherein a reducing agent is present during the extractionstep to disrupt or rearrange the disulfide bonds of trypsin inhibitorsto achieve a reduction in trypsin inhibitor activity.
 53. The process ofclaim 27 wherein a reducing agent is present during the concentrationstep and/or optional diafiltration step to disrupt or rearrange thedisulfide bonds of trypsin inhibitors to achieve a reduction in trypsininhibitor activity.
 54. The process of claim 44 wherein a reducing agentis added to the concentrated and optionally diafiltered soy proteinsolution prior to drying and/or the dried soy protein product to disruptor rearrange the disulfide bonds of trypsin inhibitors to achieve areduction in trypsin inhibitor activity.
 55. The process of claim 27wherein the concentrated and optionally diafiltered soy protein solutionis diluted to form a precipitate, the precipitate is separated from thesupernatant thereto, and the separated precipitate is dried orsolubilized in water at low pH to form a protein solution.
 56. Theprocess of claim 55 wherein the dilution is effected about 5 to about 25fold with water.
 57. The process of claim 56 wherein the water used toeffect the dilution has a temperature of about 1° to about 65° C. 58.The process of claim 55 wherein the precipitate is washed with about 1to about 10 volumes of water and then the precipitate is recovered. 59.The process of claim 55 wherein the precipitate is dried to form a soyprotein product having a protein content of at least about 60 wt %(N×6.25) d.b.
 60. The process of claim 55 wherein the precipitate issolubilized in water at a pH of about 1.5 to about 4.4 to form a soyprotein solution, which may be dried.
 61. The process of claim 60wherein the protein solution is dried to form a soy protein producthaving a protein content of at least about 60 wt % (N×6.25) d.b.
 62. Theprocess of claim 60 wherein the soy protein solution is subjected to aheat treatment step to inactivate heat labile anti-nutritional factors.63. The process of claim 60 wherein the soy protein solution isconcentrated to increase the concentration thereof while maintaining theionic strength substantially constant by using a selective membranetechnique and optionally diafiltered.
 64. The process of claim 63wherein the concentrated and optionally diafiltered soy protein solutionis treated with an adsorbent to remove colour and/or odour compounds.65. The process of claim 63 wherein the concentrated and optionallydiafiltered soy protein solution is dried to form a soy protein producthaving a protein content of at least about 60 wt % (N×6.25) d.b.
 66. Theprocess of claim 56 wherein the dilution is effected with about 10 toabout 20 fold with water.
 67. The process of claim 57 wherein the waterused to effect the dilution.
 68. The process of claim 58 wherein theprecipitate is washed with about 2 to about 3 volumes of water.
 69. Theprocess of claim 59, wherein the soy protein product has a proteincontent of at least about 90 wt % (N×6.25) d.b.
 70. The process of claim69, wherein the soy protein product has a protein content of at leastabout 100 wt % (N×6.25) d.b.
 71. The process of claim 60, wherein theprecipitate is solubilized in about 2 to about 3 volumes of water at apH of about 2.0 to about 4.0.
 72. The process of claim 61 wherein thesoy protein product has a protein content of at least about 90 wt %(N×0.6.25) d.b.
 73. The process of claim 72 wherein the soy proteinproduct has a protein content of at least about 100 wt % (N×0.6.25) d.b.74. The process of claim 65 wherein the soy protein product has aprotein content of at least about 90 wt % (N×0.6.25) d.b.
 75. Theprocess of claim 74 wherein the soy protein product has a proteincontent of at least about 100 wt % (N×0.6.25) d.b.
 76. A process ofproducing a soy protein product having a soy protein content of at leastabout 60 wt % (N×6.25) on a dry weight basis, which comprises: (a)extracting a soy protein source with an aqueous calcium salt solution ata pH of about 1.5 to about 5 to cause solubilization of soy protein fromthe soy protein source and to form an aqueous soy protein solution, (b)at least partially separating the aqueous soy protein solution fromresidual soy protein source, (c) concentrating said soy protein solutionwhile maintaining the ionic strength thereof substantially constant toproduce a concentrated soy protein solution having a proteinconcentration of about 50 to about 70 g/L and the concentrated soyprotein solution is optionally diafiltered, (d) diluting theconcentrated and optionally diafiltered soy protein solution to form aprecipitate, separating the precipitate from the supernatant thereto andeither drying the separated precipitate or solubilizing the separatedprecipitate in water at low pH to form a protein solution, and (e) pHadjusting the concentrated and optionally diafiltered soy proteinsolution prior to the dilution step to a pH of about 1.5 to about 7.0.77. The process of claim 76 wherein the pH in step (e) is about 4.0 toabout 7.0.
 78. The process of claim 77 wherein the pH in step (e) is 5.0to 7.0.
 79. A process of producing a soy protein product having a soyprotein content of at least about 60 wt % (N×6.25) on a dry weightbasis, which comprises: (a) extracting a soy protein source with anaqueous calcium salt solution at a pH of about 1.5 to about 5 to causesolubilization of soy protein from the soy protein source and to form anaqueous soy protein solution, (b) at least partially separating theaqueous soy protein solution from residual soy protein source, (c)concentrating said soy protein solution while maintaining the ionicstrength thereof substantially constant to produce a concentrated soyprotein solution having a protein concentration of about 50 to about 70g/L and the concentrated soy protein solution is optionally diafiltered,and (d) diluting the concentrated and optionally diafiltered soy proteinsolution to form a precipitate which is re-solubilized in the dilutionwater by pH adjustment to form a soy protein solution.
 80. The processof claim 79 wherein the concentrated and optionally diafiltered soyprotein solution is pH-adjusted prior to the dilution step to a pH ofabout 1.5 to about 7.0.
 81. The process of claim 80 wherein the pH isabout 4.0 to about 7.0.
 82. The process of claim 79 wherein theprecipitate is re-solubilized in the dilution water by pH adjustment ofthe mixture subsequent to the dilution step to a pH of about 1.5 toabout 4.4.
 83. The process of claim 82 wherein the pH is about 2.0 toabout 4.0.
 84. The process of claim 79 wherein the dilution is effectedabout 1 to about 25 fold with water.
 85. The process of claim 84 whereinthe water used to effect the dilution has a temperature of about 1 toabout 65° C.
 86. The process of claim 79 wherein the soy proteinsolution is dried to form a soy protein product having a protein contentof at least about 60 wt % (N×6.25) d.b.
 87. The process of claim 79wherein the soy protein solution formed in step (d) is subjected to aheat treatment step to inactivate heat labile anti-nutritional factors.88. The process of claim 79 wherein the soy protein solution isconcentrated to increase the concentration thereof while maintaining theionic strength substantially constant by using a selective membranetechnique, and optionally diafiltered.
 89. The process of claim 88wherein the concentrated and optionally diafiltered soy protein solutionis treated with an adsorbent to remove colour and/or odour compounds.90. The process of claim 81 wherein the pH is 5.0 to 7.0.
 91. Theprocess of claim 82 wherein the temperature is about 20° to about 35° C.92. The process of claim 86 wherein the soy protein product is a soyprotein solution having a protein content of at least about 90 wt %(N×6.25) d.b.
 93. The process of claim 92 wherein the soy proteinisolate has a protein content of at least about 100 wt % (N×6.25) d.b.94. The process of claim 84 wherein the dilution is effected about 3 toabout 12 fold with water.